Defining West Nile virus infection of the lymphatics using single cell techniques

ABSTRACT Mosquito- and tick-borne neurotropic arboviruses cause annual epidemics of virus-induced encephalitis throughout the world and are considered some of the most rapidly spreading vector-borne diseases. West Nile virus (WNV) is the leading cause of mosquito-borne encephalitis in humans in the United States and there are currently no antiviral therapeutics or vaccines to control or prevent infection\disease. The major focus of this proposal is to develop and use innovative single cell techniques to gain unprecedented insight into WNV infection of the lymphatics and spleen. This will allow us to better define the cellular targets of infection and understand how the antiviral response limits virus replication and spread at a single cell level. The pathogenesis of WNV in humans is incompletely defined, although excellent immunocompetent mouse models have illuminated mechanisms of virus-induced encephalitis and features of immune control. Following delivery into the skin, WNV replicates in keratinocytes and innate cells within the myeloid lineage, including dendritic cells (DCs) and macrophages. We have found that WNV quickly spreads from the nearest draining lymph node to other peripheral and CNS-draining lymph nodes (deep cervical and superficial) as well as the spleen. Eventually, this leads to a viremic phase followed by viral entry to the CNS and infection of neurons in the brain. We and others have determined that virus control early during infection, especially within the peripheral lymph nodes and spleen is essential for limiting virus dissemination into the central nervous system. The RIG-I like receptor and type I interferon (IFN) signaling pathway are critical for promoting antiviral immune responses that serve to control virus replication, dissemination to the CNS and infection outcome. High-throughput genomic technologies have been an invaluable tool for studying the host response to WNV infection over the past several years. While these technologies have revealed novel pathways and innate immune networks that control viral replication, a key limitation to these genomic studies is that they rely on measurements from bulk cell populations which does not account for heterogeneity in the cellular response to a virus infection. Single cell analysis can reveal rare cell types, distinguish between infected and uninfected bystander cells, and identify unique subpopulations important for the host response to virus infection. Our proposed study utilizes a multidisciplinary approach that combines virology, immunology, molecular biology, and systems biology to developing single cell methodologies, identify in vivo cellular targets of infection, and evaluate the host response to WNV infection within infected and bystander cells. In Aim 1, we will develop a WNV-inclusive single cell RNA sequencing method to identify and profile WNV infected and bystander cells. In Aim 2, we will identify the cellular targets of infection and molecular pathways that restrict WNV infection in the lymphatics. Understanding early events that take place after WNV infection at the single cell resolution may help guide development of targeted therapeutics and vaccines.